Biology subjects

Pletz, M. W.

Publications and source records attributed to Pletz, M. W..

6 recordsLinked to original sources

bioRxiv2024

Diversification of Pseudomonas aeruginosa biofilm populations under repeated phage exposures decreases the efficacy of the treatment

Phage therapy has been proposed as a therapeutic alternative to antibiotics for treatment of chronic, biofilm-related P. aeruginosa infections. To get a deeper insight into the complex biofilm-phage interactions, we investigated in the present study the effect of three successive exposures to lytic phages of biofilms formed by the reference strains PAO1 and PA14 as well as of two sequential clinical P. aeruginosa isolates from the sputum of a patient with cystic fibrosis (CF). The Calgary device was employed as biofilm model and the efficacy of phage treatment was evaluated by measurements of the biomass stained with crystal violet (CV) and of the cell density of the biofilm bacterial population (CFU/ml) after each of the three phage exposures. The genetic alterations of P. aeruginosa isolates from biofilms exposed to phages were investigated by whole genome sequencing. We show here that the anti-biofilm efficacy of the phage treatment decreased rapidly with repeated applications of lytic phages on P. aeruginosa strains with different genetic background. Although we observed the maintenance of a small subpopulation of sensitive cells after repeated phage treatments, a fast recruitment of mechanisms involved in the persistence of biofilms to the phage attack occurred, mainly by mutations causing alterations of the phage receptors. However, mutations causing phage tolerant phenotypes such as alginate-hyperproducing mutants were also observed. In conclusion, a decreased anti-biofilm effect occurred after repeated exposure to lytic phages of P. aeruginosa biofilms due to recruitment of different resistance and tolerance mechanisms.

microbiology↗

bioRxiv2024

Temperate bacteriophage induced in Pseudomonas aeruginosa biofilms can modulate bacteriophage and antibiotic resistance

Given the high levels of resistance in Gram-negative bacteria, phage therapy is garnering increasing attention. In Germany, a clinical study is already underway investigating a phage cocktail for the treatment of Pseudomonas aeruginosa in cystic fibrosis (CF) patients. In our study, we examined susceptibility to virulent phages and the PF1-like prophage and antimicrobial profiles and of P. aeruginosa isolates from a local cystic fibrosis cohort to identify correlations and lysogenic conversion of the prophegs. Consistent with other studies, prophage Pf4 is the most prevalent in this cohort and is activated in the absence of other influences during biofilm formation. These phages can be transferred to other strains that do not contain Pf1-like prophages, thereby influencing the dynamics of bacterial populations in the CF lung. This also rapidly leads to the emergence of a subpopulation resistant to the virulent phages, potentially complicating phage therapy. However, this subset also becomes more susceptible to most antibiotics commonly used in CF, which could be a useful treatment strategy. Interestingly, this bacterial subset lost its susceptibility to colistin, an important inhaled antibiotic in CF, which could lead to treatment failure. Our research highlights both the difficulties and potential strategies to improve treatment options for CF patients. Author summaryWe investigated 51 P. aeruginosa isolates obtained from CF patients for the presence of PF1-like prophages and characterized their susceptibility prior and after lysogenig conversion of the prophages to three virulent phages. Our study revealed that the temperate phage Pf4 is the most prominent PF1-like prophage in this cohort, undergoing lysogenic conversion during biofilm formation. The virions identified in the biofilm supernatants are superinfective and transferable to other prophage-free P. aeruginosa isolates, shaping population dynamics in the CF lung. Prophage reactivation results in the survival of a sub-population with reduced susceptibility to virulent phages, posing a potential challenge for phage therapy. However, this sub-population exhibited restored susceptibility to most CF-relevant antibiotics, presenting an intriguing therapeutic opportunity. Targeted prophage reactivation could sensitize multidrug-resistant P. aeruginosa in CF patients, enhancing or even restoring antimicrobial efficacy. Notably, this sub-population also showed a loss of colistin susceptibility, which may lead to treatment failure when colistin is used as an inhaled antipseudomonal antibiotic. Our findings highlight both significant challenges and potential therapeutic opportunities for optimizing the treatment of CF patients. However, these findings are preliminary and require further investigation, particularly regarding the possibility of lysogenic conversion in other prophages (such as Siphoviridae) and how these interplays with resistance to virulent phages. Further studies are ongoing and will enhance our understanding of the role of prophages in the pathophysiology of CF.

microbiology↗

bioRxiv2023

Nanopore sequencing for accurate bacterial outbreak tracing

Our study investigated the effectiveness of Oxford Nanopore Technologies for accurate outbreak tracing by resequencing 33 isolates of a three-year-long Klebsiella pneumoniae outbreak with Illumina short read sequencing data as the point of reference. We detected considerable base errors through cgMLST and phylogenetic analysis of genomes sequenced with Oxford Nanopore Technologies, leading to the false exclusion of some outbreak-related strains from the outbreak cluster. Nearby methylation sites cause these errors and can also be found in other species besides K. pneumoniae. Based on this data, we explored PCR-based sequencing and a masking strategy, which both successfully addressed these inaccuracies and ensured accurate outbreak tracing. We offer our masking strategy as a bioinformatic workflow (MPOA is freely available on GitHub under the GNUv3 license: github.com/replikation/MPOA) to identify and mask problematic genome positions in a reference-free manner. Our research highlights limitations in using Oxford Nanopore Technologies for sequencing prokaryotic organisms, especially for investing outbreaks. For time-critical projects that cannot wait for further technological developments by Oxford Nanopore Technologies, our study recommends either PCR-based sequencing or using our provided bioinformatic workflow. We would advise that read mapping-based quality control of genomes should be provided when publishing results.

molecular biology↗

bioRxiv2021

Evaluation of microbiome enrichment and host DNA depletion in human vaginal samples using Oxford Nanopore's adaptive sequencing

Metagenomic sequencing is promising for clinical applications to study microbial composition concerning disease or patient outcomes. Alterations of the vaginal microbiome are associated with adverse pregnancy outcomes, like preterm premature rupture of membranes and preterm birth. Methodologically these samples often have to deal with low relative amounts of prokaryotic DNA and high amounts of host DNA (> 90%), decreasing the overall microbial resolution. Nanopores adaptive sampling method offers selective DNA depletion or target enrichment to directly reject or accept DNA molecules during sequencing without specialized sample preparation. Here, we demonstrate how selective human host depletion resulted in a 1.70 fold ({+/-} 0.27 fold) increase in total sequencing depth, providing higher taxonomic profiling sensitivity. At the same time, the microbial composition remains consistent with the control experiments. The complete removal of all human host sequences is not yet possible and should be considered as an ethical approval statement might still be necessary. Adaptive sampling increased microbial sequencing yield in all 15 sequenced clinical routine vaginal samples, making it a valuable tool for clinical surveillance and medical-based research, which can be used in addition to other host depletion methods before sequencing.

molecular biology↗

bioRxiv2020

SARS-CoV-2 causes severe alveolar inflammation and barrier dysfunction

Infections with SARS-CoV-2 lead to mild to severe coronavirus disease-19 (COVID-19) with systemic symptoms. Although the viral infection originates in the respiratory system, it is unclear how the virus can overcome the alveolar barrier, which is observed in severe COVID-19 disease courses. To elucidate the viral effects on the barrier integrity and immune reactions, we used mono-cell culture systems and a complex human alveolus-on-a-chip model composed of epithelial, endothelial, and mononuclear cells. Our data show that SARS-CoV-2 efficiently infected epithelial cells with high viral loads and inflammatory response, including the interferon expression. By contrast, the adjacent endothelial layer was no infected and did neither show productive virus replication or interferon release. With prolonged infection, both cell types are damaged, and the barrier function is deteriorated, allowing the viral particles to overbear. In our study, we demonstrate that although SARS-CoV-2 is dependent on the epithelium for efficient replication, the neighboring endothelial cells are affected, e.g., by the epithelial cytokine release, which results in the damage of the alveolar barrier function and viral dissemination.

microbiology↗

bioRxiv2020

What the Phage: A scalable workflow for the identification and analysis of phage sequences

Phages are among the most abundant and diverse biological entities on earth. Phage prediction from sequence data is a crucial first step to understanding their impact on the environment. A variety of bacteriophage prediction tools have been developed over the years. They differ in algorithmic approach, results, and ease of use. We, therefore, developed "What the Phage" (WtP), an easy-to-use and parallel multitool approach for phage prediction combined with an annotation and classification downstream strategy, thus, supporting the users decision-making process by summarizing the results of the different prediction tools in charts and tables. WtP is reproducible and scales to thousands of datasets through a workflow manager (Nextflow). WtP is freely available under a GPL-3.0 license (https://github.com/replikation/What_the_Phage).

bioinformatics↗